Bottom Line:
In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa.Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated.Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism.

Affiliation: School of Biological Sciences, the University of Hong Kong, Pokfulam, Hong Kong, China. bllim@hku.hk.

ABSTRACT

Background: Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa.

Results: Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and -lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants.

Conclusions: Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive.

Figure 1: Growth phenotype of transgenic Camelina at day 40. a, All three OE lines produced more branches, grew faster, and flowered earlier than the WT and -lines. b, Expression of AtPAP2 in OE lines was confirmed by Western blotting using AtPAP2-specific antibodies. OE lines had higher expression of AtPAP2, while there was a homologous protein of AtPAP2 in Camelina.

Mentions:
Multiple independent overexpression lines of Camelina sativa with AtPAP2 protein expression were obtained through Agrobacterium transformation. After confirmation by genomic PCR and western blotting (Figure 1), three independent T2 AtPAP2 overexpression (OE) lines were selected for characterization. When there was a 3:1 segregation of T1 transgenic seeds, -lines were selected for their relatively short hypocotyl lengths on MS plates. Identities of the three -lines were confirmed by genomic PCR and western blotting (Figure 1). A weak western blot signal was observed in protein extracts from the lines and WT. We attribute this signal to likely cross-reactivity between the AtPAP2 antibody and the endogenous AtPAP2-like protein (91% amino acid sequence identity) that has been detected in transcriptome analysis of Camelina sativa leaves (unpublished data). All plants were grown on MS agar for a week and then transferred to a growth chamber (150 μmol photons/m2/s) under a 16 h light 22°C/8 h dark 18°C photoperiod/temperature regimen. The plants were transferred immediately after first flowering to greenhouse conditions at 22°C under Hong Kong's natural day/night photoperiod. Compared to WT and their respective -lines, all three AtPAP2 overexpressor lines (OE6, OE14 and OE20) grew faster and produced more lateral branches (Figure 1). Typically, plants of the three OE lines started flowering 7-9 days earlier than WT and -lines in all three generations. Only the results of the T3 generation are presented in Table 1 for statistical reason. When the WT and three -lines first flowered at an age of 40-d, AtPAP2 OE lines had already produced more branches (Figure 1).

Figure 1: Growth phenotype of transgenic Camelina at day 40. a, All three OE lines produced more branches, grew faster, and flowered earlier than the WT and -lines. b, Expression of AtPAP2 in OE lines was confirmed by Western blotting using AtPAP2-specific antibodies. OE lines had higher expression of AtPAP2, while there was a homologous protein of AtPAP2 in Camelina.

Mentions:
Multiple independent overexpression lines of Camelina sativa with AtPAP2 protein expression were obtained through Agrobacterium transformation. After confirmation by genomic PCR and western blotting (Figure 1), three independent T2 AtPAP2 overexpression (OE) lines were selected for characterization. When there was a 3:1 segregation of T1 transgenic seeds, -lines were selected for their relatively short hypocotyl lengths on MS plates. Identities of the three -lines were confirmed by genomic PCR and western blotting (Figure 1). A weak western blot signal was observed in protein extracts from the lines and WT. We attribute this signal to likely cross-reactivity between the AtPAP2 antibody and the endogenous AtPAP2-like protein (91% amino acid sequence identity) that has been detected in transcriptome analysis of Camelina sativa leaves (unpublished data). All plants were grown on MS agar for a week and then transferred to a growth chamber (150 μmol photons/m2/s) under a 16 h light 22°C/8 h dark 18°C photoperiod/temperature regimen. The plants were transferred immediately after first flowering to greenhouse conditions at 22°C under Hong Kong's natural day/night photoperiod. Compared to WT and their respective -lines, all three AtPAP2 overexpressor lines (OE6, OE14 and OE20) grew faster and produced more lateral branches (Figure 1). Typically, plants of the three OE lines started flowering 7-9 days earlier than WT and -lines in all three generations. Only the results of the T3 generation are presented in Table 1 for statistical reason. When the WT and three -lines first flowered at an age of 40-d, AtPAP2 OE lines had already produced more branches (Figure 1).

Bottom Line:
In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa.Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated.Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism.

Affiliation:
School of Biological Sciences, the University of Hong Kong, Pokfulam, Hong Kong, China. bllim@hku.hk.

ABSTRACT

Background: Lipids extracted from seeds of Camelina sativa have been successfully used as a reliable source of aviation biofuels. This biofuel is environmentally friendly because the drought resistance, frost tolerance and low fertilizer requirement of Camelina sativa allow it to grow on marginal lands. Improving the species growth and seed yield by genetic engineering is therefore a target for the biofuels industry. In Arabidopsis, overexpression of purple acid phosphatase 2 encoded by Arabidopsis (AtPAP2) promotes plant growth by modulating carbon metabolism. Overexpression lines bolt earlier and produce 50% more seeds per plant than wild type. In this study, we explored the effects of overexpressing AtPAP2 in Camelina sativa.

Results: Under controlled environmental conditions, overexpression of AtPAP2 in Camelina sativa resulted in longer hypocotyls, earlier flowering, faster growth rate, higher photosynthetic rate and stomatal conductance, increased seed yield and seed size in comparison with the wild-type line and -lines. Similar to transgenic Arabidopsis, activity of sucrose phosphate synthase in leaves of transgenic Camelina was also significantly up-regulated. Sucrose produced in photosynthetic tissues supplies the building blocks for cellulose, starch and lipids for growth and fuel for anabolic metabolism. Changes in carbon flow and sink/source activities in transgenic lines may affect floral, architectural, and reproductive traits of plants.

Conclusions: Lipids extracted from the seeds of Camelina sativa have been used as a major constituent of aviation biofuels. The improved growth rate and seed yield of transgenic Camelina under controlled environmental conditions have the potential to boost oil yield on an area basis in field conditions and thus make Camelina-based biofuels more environmentally friendly and economically attractive.